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Magnetic Resonance Imaging in the Assessment of Anti Prion Mediated Neurotoxicity In Vivo

Reimann, Regina. Magnetic Resonance Imaging in the Assessment of Anti Prion Mediated Neurotoxicity In Vivo. 2015, University of Zurich, Faculty of Medicine.

Abstract

The normal cellular prion protein (PrPc) plays a dual role in transmissible spongiform encephalopathies (TSE), which is a group of lethal disease affecting humans and a variety of animal species. First PrPC is hypothesized to be the source of the causative agent “the prion” in TSE, as the pathologic missfolded scrapie isoform of the prion protein (PrPSC) catalyses its own conversion from the prion protein. Second, there is substantial evidence that PrPC is the receptor mediating neurotoxicity and disease progression in TSE. In support of this theory, some antiprion antibodies have been found to induce neurotoxicity, an important discovery that may provide a new model system to investigate the pathologic interaction between PrPSC and PrPC.In the first part of the thesis I investigated antiprion mediated toxicity in vivo. Towards this goal, I established two read outs based on magnetic resonance imaging (MRI) volumetry allowing the measurment of antiprion induced neurotoxicity in intact mice over time. In the beginning I worked with Manganese Enhanced MRI (MEMRI) to visualize cerebellar lesion induction. As this was less well suited to the measurement of the neurotoxic induction in the hippocampus, I additionally established a diffusion weighted imaging (DWI) scan protocol. Diffusion restriction can be found within an hour after the injection of high concentration of monovalent fragments of the neurotoxic antiprion antibody POM1. As previously found in cerebellar organotypic slice cultures (COCS) antiprion mediated neurotoxicity was found to be target and eptiopic specific. Neuronal expression of PrPC is sufficient for lesion induction and lesion induction is independent from cross-linking.Further I investigated important signalling pathways downstream of PrPC. Reactive oxygen species (ROS) are a known mediator of neurodegenerative disease and I could demonstrate that they are important in the pathologic cascade of antiprion mediated neurotoxicitiy in vivo.As a major source of ROS, I identified NADPH oxidase 2 (NOX2). As in bona fide prion infection, I could detect fodrin cleavage as a marker for calpain activation in homogenates from antiprion injected brain tissue. These findings are indicative that similar pathways are activated in both pathological conditions. Further, my data shows that the NCX3 antiporter is a possible source of pathologic Ca2+ currents in the antiprion antibody model.In the second part of this thesis, I focused on the risk characterization of neurotoxic antiprion antibodies. Despite reports about the neurotoxic side effects of antiprion antibodies, passive immunotherapy with these ligands is still a therapeutic strategy under investigation in the treatment of TSE. Using my established MRI based quantification system and basic histological methods, I assessed the neurotoxic potential of the antiprion antibody ICSM18, which is under evaluation as a therapeutic agent for TSE in humans. Unless further investigations can confirm a safe therapeutic window for the use of these antibodies and others, my findings suggest that utmost caution is indicated.Here I show that Magnetic Resonance Imaging is a valuable tool in the assessment of anti prion mediated neurotoxicity in vivo. This technique can be used for the risk characterization of antiption antibodies. In addition, our work in vivo and in cerebellar slice cultures providesnew evidence that neurotoxic antiprion antibodies model the pathologic interaction of PrPSC with PrPC. Thus, the established tool can be used in further studies to investigate prionmediated neurotoxicity, in a much shorter time frame and within biosafety level one.

The normal cellular prion protein (PrPc) plays a dual role in transmissible spongiform encephalopathies (TSE), which is a group of lethal disease affecting humans and a variety of animal species. First PrPC is hypothesized to be the source of the causative agent “the prion” in TSE, as the pathologic missfolded scrapie isoform of the prion protein (PrPSC) catalyses its own conversion from the prion protein. Second, there is substantial evidence that PrPC is the receptor mediating neurotoxicity and disease progression in TSE. In support of this theory, some antiprion antibodies have been found to induce neurotoxicity, an important discovery that may provide a new model system to investigate the pathologic interaction between PrPSC and PrPC.In the first part of the thesis I investigated antiprion mediated toxicity in vivo. Towards this goal, I established two read outs based on magnetic resonance imaging (MRI) volumetry allowing the measurment of antiprion induced neurotoxicity in intact mice over time. In the beginning I worked with Manganese Enhanced MRI (MEMRI) to visualize cerebellar lesion induction. As this was less well suited to the measurement of the neurotoxic induction in the hippocampus, I additionally established a diffusion weighted imaging (DWI) scan protocol. Diffusion restriction can be found within an hour after the injection of high concentration of monovalent fragments of the neurotoxic antiprion antibody POM1. As previously found in cerebellar organotypic slice cultures (COCS) antiprion mediated neurotoxicity was found to be target and eptiopic specific. Neuronal expression of PrPC is sufficient for lesion induction and lesion induction is independent from cross-linking.Further I investigated important signalling pathways downstream of PrPC. Reactive oxygen species (ROS) are a known mediator of neurodegenerative disease and I could demonstrate that they are important in the pathologic cascade of antiprion mediated neurotoxicitiy in vivo.As a major source of ROS, I identified NADPH oxidase 2 (NOX2). As in bona fide prion infection, I could detect fodrin cleavage as a marker for calpain activation in homogenates from antiprion injected brain tissue. These findings are indicative that similar pathways are activated in both pathological conditions. Further, my data shows that the NCX3 antiporter is a possible source of pathologic Ca2+ currents in the antiprion antibody model.In the second part of this thesis, I focused on the risk characterization of neurotoxic antiprion antibodies. Despite reports about the neurotoxic side effects of antiprion antibodies, passive immunotherapy with these ligands is still a therapeutic strategy under investigation in the treatment of TSE. Using my established MRI based quantification system and basic histological methods, I assessed the neurotoxic potential of the antiprion antibody ICSM18, which is under evaluation as a therapeutic agent for TSE in humans. Unless further investigations can confirm a safe therapeutic window for the use of these antibodies and others, my findings suggest that utmost caution is indicated.Here I show that Magnetic Resonance Imaging is a valuable tool in the assessment of anti prion mediated neurotoxicity in vivo. This technique can be used for the risk characterization of antiption antibodies. In addition, our work in vivo and in cerebellar slice cultures providesnew evidence that neurotoxic antiprion antibodies model the pathologic interaction of PrPSC with PrPC. Thus, the established tool can be used in further studies to investigate prionmediated neurotoxicity, in a much shorter time frame and within biosafety level one.

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